ABSTRACT
BACKGROUND: Asthma is a complex disease with multiple phenotypes that may differ in disease pathobiology and treatment response. IL33 single nucleotide polymorphisms (SNPs) have been reproducibly associated with asthma. IL33 levels are elevated in sputum and bronchial biopsies of patients with asthma. The functional consequences of IL33 asthma SNPs remain unknown. OBJECTIVE: This study sought to determine whether IL33 SNPs associate with asthma-related phenotypes and with IL33 expression in lung or bronchial epithelium. This study investigated the effect of increased IL33 expression on human bronchial epithelial cell (HBEC) function. METHODS: Association between IL33 SNPs (Chr9: 5,815,786-6,657,983) and asthma phenotypes (Lifelines/DAG [Dutch Asthma GWAS]/GASP [Genetics of Asthma Severity & Phenotypes] cohorts) and between SNPs and expression (lung tissue, bronchial brushes, HBECs) was done using regression modeling. Lentiviral overexpression was used to study IL33 effects on HBECs. RESULTS: We found that 161 SNPs spanning the IL33 region associated with 1 or more asthma phenotypes after correction for multiple testing. We report a main independent signal tagged by rs992969 associating with blood eosinophil levels, asthma, and eosinophilic asthma. A second, independent signal tagged by rs4008366 presented modest association with eosinophilic asthma. Neither signal associated with FEV1, FEV1/forced vital capacity, atopy, and age of asthma onset. The 2 IL33 signals are expression quantitative loci in bronchial brushes and cultured HBECs, but not in lung tissue. IL33 overexpression in vitro resulted in reduced viability and reactive oxygen species-capturing of HBECs, without influencing epithelial cell count, metabolic activity, or barrier function. CONCLUSIONS: We identify IL33 as an epithelial susceptibility gene for eosinophilia and asthma, provide mechanistic insight, and implicate targeting of the IL33 pathway specifically in eosinophilic asthma.
Subject(s)
Asthma , Gene Expression Regulation/immunology , Genetic Predisposition to Disease , Interleukin-33 , Polymorphism, Single Nucleotide , Adult , Asthma/genetics , Asthma/immunology , Female , Genome-Wide Association Study , Humans , Interleukin-33/genetics , Interleukin-33/immunology , Male , Middle AgedABSTRACT
Genome wide association (GWA) studies have reproducibly identified signals on chromosome 4q24 associated with lung function and COPD. GSTCD (Glutathione S-transferase C-terminal domain containing) represents a candidate causal gene in this locus, however little is currently known about the function of this protein. We set out to further our understanding of the role of GSTCD in cell functions and homeostasis using multiple molecular and cellular approaches in airway relevant cells. Recombinant expression of human GSTCD in conjunction with a GST activity assay did not identify any enzymatic activity for two GSTCD isoforms questioning the assignment of this protein to this family of enzymes. Protein structure analyses identified a potential methyltransferase domain contained within GSTCD, with these enzymes linked to cell viability and apoptosis. Targeted knockdown (siRNA) of GSTCD in bronchial epithelial cells identified a role for GSTCD in cell viability as proliferation rates were not altered. To provide greater insight we completed transcriptomic analyses on cells with GSTCD expression knocked down and identified several differentially expressed genes including those implicated in airway biology; fibrosis e.g. TGFBR1 and inflammation e.g. IL6R. Pathway based transcriptomic analyses identified an over-representation of genes related to adipogenesis which may suggest additional functions for GSTCD. These findings identify potential additional functions for GSTCD in the context of airway biology beyond the hypothesised GST activity and warrant further investigation.
Subject(s)
Genome-Wide Association Study/methods , Homeostasis/physiology , Lung/physiology , Myocytes, Smooth Muscle/physiology , Proteins/genetics , Respiratory Mucosa/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Humans , Lung/cytology , Proteins/metabolism , Respiratory Mucosa/cytologyABSTRACT
BACKGROUND: Meta-analyses of genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) spanning the 5-hydroxytryptamine receptor 4 (5-HT4R) gene (HTR4) associated with lung function. The aims of this study were to i) investigate the expression profile of HTR4 in adult and fetal lung tissue and cultured airway cells, ii) further define HTR4 gene structure and iii) explore the potential functional implications of key SNPs using a bioinformatic approach. METHODS: Following reverse transcription (RT)-PCR in human brain, 5' rapid amplification of cDNA ends (5' RACE) was used to examine the exonic structure of HTR4 at the 5' end. Quantitative (Q)-PCR was used to quantify HTR4 mRNA expression in total RNA from cultured airway cells and whole lung tissue. Publically available gene microarray data on fetal samples of estimated gestational age 7-22 weeks were mined for HTR4 expression. Immunohistochemistry (IHC; in adult and fetal lung tissue) and a radioligand binding assay (in cultured airway cells) were used to analyze 5-HT4R protein expression. RESULTS: IHC in adult lung, irrespective of the presence of chronic obstructive pulmonary disease (COPD), suggested low level expression of 5-HT4R protein, which was most prominent in alveolar pneumocytes. There was evidence of differential 5-HT4R protein levels during gestation in fetal lung, which was also evident in gene expression microarray data. HTR4 mRNA expression, assessed by Q-PCR, was <0.5% relative to brain in total adult lung tissue and in human airway smooth muscle (HASM) and bronchial epithelial cells (HBEC) derived from adult donors. Radioligand binding experiments also indicated that HBEC and HASM cells did not express a significant 5-HT4R population. 5' RACE in brain identified a novel N-terminal variant, containing an extended N-terminal sequence. The functional significance of key HTR4 SNPs was investigated using the encyclopedia of DNA elements consortium (ENCODE) dataset. These analyses identified multiple alterations in regulatory motifs for transcription factors implicated in lung development, including Foxp1. CONCLUSIONS: Taken together, these data suggest a role for HTR4 in lung development, which may at least in part explain the genetic association with lung function.
Subject(s)
Gene Expression Regulation, Developmental/genetics , Lung/embryology , Lung/physiology , Receptors, Serotonin/genetics , Female , Humans , MaleABSTRACT
RATIONALE: Genome-wide association studies (GWAS) have identified loci influencing lung function, but fewer genes influencing chronic obstructive pulmonary disease (COPD) are known. OBJECTIVES: Perform meta-analyses of GWAS for airflow obstruction, a key pathophysiologic characteristic of COPD assessed by spirometry, in population-based cohorts examining all participants, ever smokers, never smokers, asthma-free participants, and more severe cases. METHODS: Fifteen cohorts were studied for discovery (3,368 affected; 29,507 unaffected), and a population-based family study and a meta-analysis of case-control studies were used for replication and regional follow-up (3,837 cases; 4,479 control subjects). Airflow obstruction was defined as FEV(1) and its ratio to FVC (FEV(1)/FVC) both less than their respective lower limits of normal as determined by published reference equations. MEASUREMENTS AND MAIN RESULTS: The discovery meta-analyses identified one region on chromosome 15q25.1 meeting genome-wide significance in ever smokers that includes AGPHD1, IREB2, and CHRNA5/CHRNA3 genes. The region was also modestly associated among never smokers. Gene expression studies confirmed the presence of CHRNA5/3 in lung, airway smooth muscle, and bronchial epithelial cells. A single-nucleotide polymorphism in HTR4, a gene previously related to FEV(1)/FVC, achieved genome-wide statistical significance in combined meta-analysis. Top single-nucleotide polymorphisms in ADAM19, RARB, PPAP2B, and ADAMTS19 were nominally replicated in the COPD meta-analysis. CONCLUSIONS: These results suggest an important role for the CHRNA5/3 region as a genetic risk factor for airflow obstruction that may be independent of smoking and implicate the HTR4 gene in the etiology of airflow obstruction.
Subject(s)
Genome-Wide Association Study , Nerve Tissue Proteins/genetics , Pulmonary Disease, Chronic Obstructive/genetics , Receptors, Nicotinic/genetics , Receptors, Serotonin, 5-HT4/genetics , Aged , Female , Forced Expiratory Volume/genetics , Humans , Male , Middle Aged , Polymorphism, Single Nucleotide/genetics , Smoking/genetics , Vital Capacity/geneticsABSTRACT
BACKGROUND: Several studies have suggested that chromosome 19q13.1-3 contains asthma susceptibility genes. OBJECTIVE: Linkage and association analyses using 587 United Kingdom and Dutch asthma families (n = 2819 subjects) were used to investigate this region. METHODS: A 3-phase procedure was used: (1) linkage and association analyses using 15 microsatellite markers spanning 14.4 mega base pairs (Mbps) on 19q13, (2) fine mapping of the refined region using 26 haplotype tagging single nucleotide polymorphisms (SNPs), and (3) dense gene analyses using 18 SNPs evaluated for association with asthma, bronchial hyperresponsiveness (BHR), FEV1, plasma urokinase plasminogen activator receptor (PLAUR), and rate of annual FEV1 decline in subjects with asthma. RESULTS: The microsatellite analyses provided tentative support for an asthma/lung function susceptibility locus (48.9-49.1Mbps), and fine mapping localized modest association to the PLAUR gene. PLAUR SNPs in the 5' region, intron 3, and 3' region are associated with asthma and BHR susceptibility and predict FEV1 and plasma PLAUR levels. SNPs in the 5' region showed association for asthma (2 populations), FEV1 (2 populations), and BHR (2 populations) phenotypes. SNPs in intron 3 showed association with asthma (2 populations) and BHR (3 populations). Importantly, the same 5' region and intron 3 SNPs were associated with plasma PLAUR levels. The same 5' region and 3' region SNPs were found to be determinants of FEV1 decline in subjects with asthma. CONCLUSION: This study represents the first report to identify PLAUR as a potential asthma susceptibility gene and determine PLAUR regions underlying this association, including a role in influencing plasma PLAUR levels. Finally, the association of PLAUR with lung function decline supports a role for PLAUR in airway remodeling in asthma.
Subject(s)
Asthma/genetics , Asthma/physiopathology , Bronchial Hyperreactivity/physiopathology , Genetic Predisposition to Disease , Receptors, Urokinase Plasminogen Activator/blood , Receptors, Urokinase Plasminogen Activator/genetics , Adolescent , Adult , Alleles , Asthma/blood , Child , Child, Preschool , Female , Gene Frequency , Haplotypes/genetics , Humans , Lung/physiopathology , Male , Microsatellite Repeats/genetics , Middle Aged , Polymorphism, Single Nucleotide , Young AdultABSTRACT
Rationale: Exposure to biomass smoke is believed to increase the risk of developing chronic obstructive pulmonary disease. However, little is known about the mechanisms underlying responses to biomass smoke in human lungs.Objectives: This study had two objectives: first, to quantify "real-life" exposures to particulate matter <2 µm in diameter (PM2.5) and carbon monoxide (CO) measured during cooking on stoves in rural areas of Nepal in different geographical settings; and second, to assess the effect of biomass smoke extracts on inflammatory responses in ex vivo human lung tissue.Methods: Personal exposures to PM2.5 and indoor near-stove CO concentrations were measured during cooking on a range of stoves in 103 households in 4 different Nepalese villages situated at altitudes between â¼100 and 4,000 m above sea level. Inflammatory profiles to smoke extracts collected in the field were assessed by incubating extracts with human lung tissue fragments and subsequent Luminex analysis.Results: In households using traditional cooking stoves, the overall mean personal exposure to PM2.5 during cooking was 276.1 µg/m3 (standard deviation [SD], 265 µg/m3), and indoor CO concentration was 16.3 ppm (SD, 19.65 ppm). The overall mean PM2.5 exposure was reduced by 51% (P = 0.04) in households using biomass fuel in improved cook stoves, and 80% (P < 0.0001) in households using liquefied petroleum gas. Similarly, the indoor CO concentration was reduced by 72% (P < 0.001) and 86% (P < 0.0001) in households using improved cook stoves and liquefied petroleum gas, respectively. Significant increases occurred in 7 of the 17 analytes measured after biomass smoke extract stimulation of human lung tissue (IL-8 [interleukin-8], IL-6, TNF-α [tumor necrosis factor-α], IL-1ß, CCL2, CCL3, and CCL13).Conclusions: High levels of real-life exposures to PM2.5 and CO occur during cooking events in rural Nepal. These exposures induce lung inflammation ex vivo, which may partially explain the increased risk of chronic obstructive pulmonary disease in these communities.
Subject(s)
Air Pollution, Indoor/analysis , Carbon Monoxide/analysis , Cooking/instrumentation , Cytokines/analysis , Inhalation Exposure/analysis , Smoke , Biomass , Environmental Monitoring/methods , Humans , Inflammation/chemically induced , Lung/pathology , Nepal , Particulate Matter/analysis , Rural PopulationABSTRACT
The IL1RL1 (ST2) gene locus is robustly associated with asthma; however, the contribution of single nucleotide polymorphisms (SNPs) in this locus to specific asthma subtypes and the functional mechanisms underlying these associations remain to be defined. We tested for association between IL1RL1 region SNPs and characteristics of asthma as defined by clinical and immunological measures and addressed functional effects of these genetic variants in lung tissue and airway epithelium. Utilizing 4 independent cohorts (Lifelines, Dutch Asthma GWAS [DAG], Genetics of Asthma Severity and Phenotypes [GASP], and Manchester Asthma and Allergy Study [MAAS]) and resequencing data, we identified 3 key signals associated with asthma features. Investigations in lung tissue and primary bronchial epithelial cells identified context-dependent relationships between the signals and IL1RL1 mRNA and soluble protein expression. This was also observed for asthma-associated IL1RL1 nonsynonymous coding TIR domain SNPs. Bronchial epithelial cell cultures from asthma patients, exposed to exacerbation-relevant stimulations, revealed modulatory effects for all 4 signals on IL1RL1 mRNA and/or protein expression, suggesting SNP-environment interactions. The IL1RL1 TIR signaling domain haplotype affected IL-33-driven NF-κB signaling, while not interfering with TLR signaling. In summary, we identify that IL1RL1 genetic signals potentially contribute to severe and eosinophilic phenotypes in asthma, as well as provide initial mechanistic insight, including genetic regulation of IL1RL1 isoform expression and receptor signaling.
Subject(s)
Asthma/genetics , Genetic Predisposition to Disease/genetics , Interleukin-1 Receptor-Like 1 Protein/genetics , Asthma/immunology , Genotype , Humans , Lung/immunology , Phenotype , Polymorphism, Single Nucleotide , Respiratory Mucosa/immunologyABSTRACT
INTRODUCTION: Genome-Wide Association Studies suggest glutathione S transferase C terminal domain (GSTCD) may play a role in development of Chronic Obstructive Pulmonary Disease. We aimed to define the potential role of GSTCD in airway inflammation and contraction using precision cut lung slice (PCLS) from wild-type (GSTCD+/+) and GSTCD knockout mice (GSTCD-/-). METHODS: PCLS from age and gender matched GSTCD+/+ and GSTCD-/- mice were prepared using a microtome. Contraction was studied after applying either a single dose of Methacholine (Mch) (1 µM) or different doses of Mch (0.001 to 100 µM). Each slice was then treated with lipopolysaccharide (LPS) or vehicle (PBS) for 24 hours. PCLS contraction in the same airway was repeated before and after stimulation. Levels of TNFα production was also measured. RESULTS: There were no differences in contraction of PCLS from GSTCD+/+ and GSTCD-/- mice in response to Mch (EC50 of GSTCD+/+ vs GSTCD-/- animals: 100.0±20.7 vs 107.7±24.5 nM, p = 0.855, n = 6 animals/group). However, after LPS treatment, there was a 31.6% reduction in contraction in the GSTCD-/- group (p = 0.023, n = 6 animals). There was no significant difference between PBS and LPS treatment groups in GSTCD+/+ animals. We observed a significant increase in TNFα production induced by LPS in GSTCD-/- lung slices compared to the GSTCD+/+ LPS treated slices. CONCLUSION: GSTCD knockout mice showed an increased responsiveness to LPS (as determined by TNFα production) that was accompanied by a reduced contraction of small airways in PCLS. These data highlight an unrecognised potential function of GSTCD in mediating inflammatory signals that affect airway responses.
Subject(s)
Bronchioles/physiology , Glutathione Transferase/genetics , Lipopolysaccharides/adverse effects , Methacholine Chloride/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Animals , Bronchioles/drug effects , Bronchioles/immunology , Disease Models, Animal , Female , Gene Knockout Techniques , Male , Mice , Muscle Contraction/drug effects , Up-RegulationABSTRACT
BACKGROUND: Few genetic studies that focus on moderate-to-severe asthma exist. We aimed to identity novel genetic variants associated with moderate-to-severe asthma, see whether previously identified genetic variants for all types of asthma contribute to moderate-to-severe asthma, and provide novel mechanistic insights using expression analyses in patients with asthma. METHODS: In this genome-wide association study, we used a two-stage case-control design. In stage 1, we genotyped patient-level data from two UK cohorts (the Genetics of Asthma Severity and Phenotypes [GASP] initiative and the Unbiased BIOmarkers in PREDiction of respiratory disease outcomes [U-BIOPRED] project) and used data from the UK Biobank to collect patient-level genomic data for cases and controls of European ancestry in a 1:5 ratio. Cases were defined as having moderate-to-severe asthma if they were taking appropriate medication or had been diagnosed by a doctor. Controls were defined as not having asthma, rhinitis, eczema, allergy, emphysema, or chronic bronchitis as diagnosed by a doctor. For stage 2, an independent cohort of cases and controls (1:5) was selected from the UK Biobank only, with no overlap with stage 1 samples. In stage 1 we undertook a genome-wide association study of moderate-to-severe asthma, and in stage 2 we followed up independent variants that reached the significance threshold of p less than 1â×â10-6 in stage 1. We set genome-wide significance at p less than 5â×â10-8. For novel signals, we investigated their effect on all types of asthma (mild, moderate, and severe). For all signals meeting genome-wide significance, we investigated their effect on gene expression in patients with asthma and controls. FINDINGS: We included 5135 cases and 25â675 controls for stage 1, and 5414 cases and 21â471 controls for stage 2. We identified 24 genome-wide significant signals of association with moderate-to-severe asthma, including several signals in innate or adaptive immune-response genes. Three novel signals were identified: rs10905284 in GATA3 (coded allele A, odds ratio [OR] 0·90, 95% CI 0·88-0·93; p=1·76â×â10-10), rs11603634 in the MUC5AC region (coded allele G, OR 1·09, 1·06-1·12; p=2·32â×â10-8), and rs560026225 near KIAA1109 (coded allele GATT, OR 1·12, 1·08-1·16; p=3·06â×â10-9). The MUC5AC signal was not associated with asthma when analyses included mild asthma. The rs11603634 G allele was associated with increased expression of MUC5AC mRNA in bronchial epithelial brush samples via proxy SNP rs11602802; (p=2·50â×â10-5) and MUC5AC mRNA was increased in bronchial epithelial samples from patients with severe asthma (in two independent analyses, p=0·039 and p=0·022). INTERPRETATION: We found substantial shared genetic architecture between mild and moderate-to-severe asthma. We also report for the first time genetic variants associated with the risk of developing moderate-to-severe asthma that regulate mucin production. Finally, we identify candidate causal genes in these loci and provide increased insight into this difficult to treat population. FUNDING: Asthma UK, AirPROM, U-BIOPRED, UK Medical Research Council, and Rosetrees Trust.
Subject(s)
Asthma/genetics , GATA3 Transcription Factor/genetics , Genetic Predisposition to Disease , Mucin 5AC , Proteins , Adult , Aged , Case-Control Studies , Female , Genome-Wide Association Study , Genotype , Humans , Male , Middle Aged , Severity of Illness Index , White PeopleABSTRACT
Chloride channels are known to play critical physiological roles in many cell types. Here, we describe the expression of anion channels using RNA Seq in primary cultures of human bronchial epithelial cells (hBECs). Chloride intracellular channel (CLIC) family members were the most abundant chloride channel transcripts, and CLIC1 showed the highest level of expression. In addition, we characterize the chloride currents in hBECs and determine how inhibition of CLIC1 via pharmacological and molecular approaches impacts these. We demonstrate that CLIC1 is able to modulate cyclic AMP-induced chloride currents and suggest that CLIC1 modulation could be important for chloride homeostasis in this cell type.
Subject(s)
Chloride Channels/metabolism , Cyclic AMP/metabolism , Respiratory Mucosa/metabolism , Bronchi/metabolism , HumansABSTRACT
Thiazide diuretics are among the most widely used treatments for hypertension, but thiazide-induced hyponatremia (TIH), a clinically significant adverse effect, is poorly understood. Here, we have studied the phenotypic and genetic characteristics of patients hospitalized with TIH. In a cohort of 109 TIH patients, those with severe TIH displayed an extended phenotype of intravascular volume expansion, increased free water reabsorption, urinary prostaglandin E2 excretion, and reduced excretion of serum chloride, magnesium, zinc, and antidiuretic hormone. GWAS in a separate cohort of 48 TIH patients and 2,922 controls from the 1958 British birth cohort identified an additional 14 regions associated with TIH. We identified a suggestive association with a variant in SLCO2A1, which encodes a prostaglandin transporter in the distal nephron. Resequencing of SLCO2A1 revealed a nonsynonymous variant, rs34550074 (p.A396T), and association with this SNP was replicated in a second cohort of TIH cases. TIH patients with the p.A396T variant demonstrated increased urinary excretion of prostaglandin E2 and metabolites. Moreover, the SLCO2A1 phospho-mimic p.A396E showed loss of transporter function in vitro. These findings indicate that the phenotype of TIH involves a more extensive metabolic derangement than previously recognized. We propose one mechanism underlying TIH development in a subgroup of patients in which SLCO2A1 regulation is altered.
Subject(s)
Hyponatremia/chemically induced , Sodium Chloride Symporter Inhibitors/adverse effects , Thiazides/adverse effects , Aged , Aged, 80 and over , Aquaporin 1/genetics , Aquaporin 2/genetics , Cohort Studies , Dinoprostone/metabolism , Female , Genome-Wide Association Study , Humans , Hyponatremia/genetics , Male , Middle Aged , Nephrons/metabolism , Organic Anion Transporters/genetics , Pharmacogenetics , Phenotype , Polymorphism, Single Nucleotide , Prostaglandins/metabolism , United Kingdom , Water/chemistryABSTRACT
Chronic obstructive pulmonary disease (COPD) is characterized by reduced lung function and is the third leading cause of death globally. Through genome-wide association discovery in 48,943 individuals, selected from extremes of the lung function distribution in UK Biobank, and follow-up in 95,375 individuals, we increased the yield of independent signals for lung function from 54 to 97. A genetic risk score was associated with COPD susceptibility (odds ratio per 1 s.d. of the risk score (â¼6 alleles) (95% confidence interval) = 1.24 (1.20-1.27), P = 5.05 × 10-49), and we observed a 3.7-fold difference in COPD risk between individuals in the highest and lowest genetic risk score deciles in UK Biobank. The 97 signals show enrichment in genes for development, elastic fibers and epigenetic regulation pathways. We highlight targets for drugs and compounds in development for COPD and asthma (genes in the inositol phosphate metabolism pathway and CHRM3) and describe targets for potential drug repositioning from other clinical indications.
Subject(s)
Genetic Loci/genetics , Genetic Predisposition to Disease/genetics , Lung/physiopathology , Pulmonary Disease, Chronic Obstructive/genetics , Adult , Aged , Aged, 80 and over , Alleles , Asthma/genetics , Epigenesis, Genetic/genetics , Female , Genome-Wide Association Study/methods , Humans , Male , Middle Aged , Polymorphism, Single Nucleotide/genetics , Risk FactorsABSTRACT
INTRODUCTION: Genome-Wide Association Studies have identified associations between lung function measures and Chronic Obstructive Pulmonary Disease (COPD) and chromosome region 6p21 containing the gene for the Advanced Glycation End Product Receptor (AGER, encoding RAGE). We aimed to (i) characterise RAGE expression in the lung, (ii) identify AGER transcripts, (iii) ascertain if SNP rs2070600 (Gly82Ser C/T) is associated with lung function and serum sRAGE levels and (iv) identify whether the Gly82Ser variant is functionally important in altering sRAGE levels in an airway epithelial cell model. METHODS: Immunohistochemistry was used to identify RAGE protein expression in 26 human tissues and qPCR was used to quantify AGER mRNA in lung cells. Gene expression array data was used to identify AGER expression during lung development in 38 fetal lung samples. RNA-Seq was used to identify AGER transcripts in lung cells. sRAGE levels were assessed in cells and patient serum by ELISA. BEAS2B-R1 cells were transfected to overexpress RAGE protein with either the Gly82 or Ser82 variant and sRAGE levels identified. RESULTS: Immunohistochemical assessment of 6 adult lung samples identified high RAGE expression in the alveoli of healthy adults and individuals with COPD. AGER/RAGE expression increased across developmental stages in human fetal lung at both the mRNA (38 samples) and protein levels (20 samples). Extensive AGER splicing was identified. The rs2070600T (Ser82) allele is associated with higher FEV1, FEV1/FVC and lower serum sRAGE levels in UK smokers. Using an airway epithelium model overexpressing the Gly82 or Ser82 variants we found that HMGB1 activation of the RAGE-Ser82 receptor results in lower sRAGE production. CONCLUSIONS: This study provides new information regarding the expression profile and potential role of RAGE in the human lung and shows a functional role of the Gly82Ser variant. These findings advance our understanding of the potential mechanisms underlying COPD particularly for carriers of this AGER polymorphism.
Subject(s)
Lung/metabolism , Pulmonary Disease, Chronic Obstructive/genetics , Receptor for Advanced Glycation End Products/genetics , Smoking , Alleles , Bronchi/cytology , Bronchi/metabolism , Case-Control Studies , Cell Line , Epithelial Cells/cytology , Epithelial Cells/metabolism , Fetus/metabolism , Genome-Wide Association Study , Genotype , Humans , Lung/pathology , Male , Middle Aged , Plasmids/genetics , Plasmids/metabolism , Polymorphism, Single Nucleotide , Pulmonary Disease, Chronic Obstructive/diagnosis , RNA Splicing , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Receptor for Advanced Glycation End Products/blood , Receptor for Advanced Glycation End Products/metabolism , Young AdultABSTRACT
Genome-Wide Association Study (GWAS) meta-analyses have identified a strong association signal for lung function, which maps to a region on 4q24 containing two oppositely transcribed genes: glutathione S-transferase, C-terminal domain containing (GSTCD) and integrator complex subunit 12 (INTS12). Both genes were found to be expressed in a range of human airway cell types. The promoter regions and transcription start sites were determined in mRNA from human lung and a novel splice variant was identified for each gene. We obtained the following evidence for GSTCD and INTS12 co-regulation and expression: (i) correlated mRNA expression was observed both via Q-PCR and in a lung expression quantitative trait loci (eQTL) study, (ii) induction of both GSTCD and INTS12 mRNA expression in human airway smooth muscle cells was seen in response to TGFß1, (iii) a lung eQTL study revealed that both GSTCD and INTS12 mRNA levels positively correlate with percent predicted FEV1, and (iv) FEV1 GWAS associated SNPs in 4q24 were found to act as an eQTL for INTS12 in a number of tissues. In fixed sections of human lung tissue, GSTCD protein expression was ubiquitous, whereas INTS12 expression was predominantly in epithelial cells and pneumocytes. During human fetal lung development, GSTCD protein expression was observed to be highest at the earlier pseudoglandular stage (10-12 weeks) compared with the later canalicular stage (17-19 weeks), whereas INTS12 expression levels did not alter throughout these stages. Knowledge of the transcriptional and translational regulation and expression of GSTCD and INTS12 provides important insights into the potential role of these genes in determining lung function. Future work is warranted to fully define the functions of INTS12 and GSTCD.
Subject(s)
Carrier Proteins/genetics , Gene Expression Regulation , Glutathione Transferase/genetics , Lung/metabolism , Proteins/genetics , Carrier Proteins/metabolism , Forced Expiratory Volume/drug effects , Forced Expiratory Volume/genetics , Gene Expression Regulation/drug effects , Gene Rearrangement/genetics , Genetic Loci/genetics , Genome, Human/genetics , Glutathione Transferase/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Lung/cytology , Lung/embryology , Lung/physiology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Nucleotide Motifs/genetics , Polymerase Chain Reaction , Proteins/metabolism , Quantitative Trait Loci/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, DNA , Transforming Growth Factor beta1/pharmacologyABSTRACT
A sequence variant (rs7216389-T) near the ORMDL3 gene on chromosome 17q21 was recently found to be associated with childhood asthma. We sought to evaluate the effect of rs7216389-T on asthma subphenotypes and its correlation with expression levels of neighboring genes. The association of rs7216389-T with asthma was replicated in six European and one Asian study cohort (N=4917 cases N=34 589 controls). In addition, we found that the association of rs7216389-T was confined to cases with early onset of asthma, particularly in early childhood (age: 0-5 years OR=1.51, P=6.89.10(-9)) and adolescence (age: 14-17 years OR=1.71, P=5.47.10(-9)). A weaker association was observed for onset between 6 and 13 years of age (OR=1.17, P=0.035), but none for adult-onset asthma (OR=1.07, P=0.12). Cases were further stratified by sex, asthma severity and atopy status. An association with greater asthma severity was observed among early-onset asthma cases (P=0.0012), but no association with sex or atopy status was observed among the asthma cases. An association between sequence variants and the expression of genes in the 17q21 region was assessed in white blood cell RNA samples collected from Icelandic individuals (n=743). rs7216389 associated with the expression of GSDMB and ORMDL3 genes. However, other sequence variants showing a weaker association with asthma compared with that of rs7216389 were more strongly associated with the expression of both genes. Thus, the contribution of rs7216389-T to the development of asthma is unlikely to operate only through an impact on the expression of ORMDL3 or GSDMB genes.